Capillary electrophoresis (CE) has been established as an important separation technique in bioanalytical chemistry. Separation and detection of very small amounts of biological samples, about pL-nL volumes, can be achieved with CE. This is generally not possible with more conventional separatory methods, even high-performance liquid chromatography (HPLC). There are several CE separation modes in use for different kinds of samples. They include capillary zone electrophoresis, moving boundary capillary electrophoresis, capillary isotachophoresis and capillary isoelectric focusing (cIEF).
CE provides high-resolution and high efficiency separation and is used in proteomic research and biopharmaceutical applications. Not only proven as a powerful analytical tool, CE is also promising for application in nano and micro-fractionation collection. For instance, on-line coupling of CE with mass spectrometry (MS) is used to elucidate protein structure, and off-line CE fractionation collection is important for further characterization of proteins in connection with sequencing, peptide digesting and mapping and reaction studies.
Various designs of microfluidic apparatus have been used for CE fraction collection. Vial collection and membrane collection of individual components of an analyte at the exit of the separation column, with or without the help of sheath fluid, has been investigated. In point-detection capillary electrophoresis, sample fractions are collected from the outlet of the separation channel after passing the detection window. Karger et al. [1] further developed vial collection to a fraction collector. As CE is normally run with 25 to 75 μm i.d. capillaries or micro-channels, extremely small volumes of individual fractions can be expected. Therefore, exact timing is important for precise fractionation. Cross-contamination frequently occurs for closely migrating peaks due to the extremely narrow peak width and extremely small amount of eluant.
An important CE mode, capillary isoelectric focusing (cIEF), is used for separating amphoteric substances such as peptides and proteins in a capillary or micro-channel under an electric field. Across the separation capillary or channel, voltage is applied and a pH gradient is created by carrier ampholytes that have been pre-mixed with the analyte sample, acidic at the anodic end of the channel and alkaline at the cathodic end of the channel. Each component in the analyte mixture migrates to a position in the separation channel where the surrounding pH corresponds to its isoelectric point. Therein, as zwitterions possessing no net charge, molecules of that component cease to move in the electric field. Different amphoteric components are thereby focused into narrow, stationary zones.
cIEF is the highest resolution CE mode for charge-based separation of amphoteric substances such as proteins and peptides. It has most often been used to separate closely related proteins having subtle differences between their structures. In the cIEF separation channel, components of the analyte mixture, which evenly distribute along the whole channel before the separation process, are separated and focused into narrow, stationary, component zones.
Uniquely narrow zones are formed using cIEF because: 1) zone broadening due to parabolic flow is not a factor in the separation process; 2) the focusing force is reverse to diffusion and 3) the electrophoresis current during focusing is low compared to other CE modes thus minimizing the effects of component zone broadening due to Joule heating. For these reasons, the analyte components from cIEF are concentrated over a hundredfold in their separated, narrow zones.
In column or micro-channel separation technologies, the narrower the component zone is the higher the resolution. Narrow zones in other CE modes can be achieved by injecting a very small ‘analyte plug’ representing a very small segment of the whole separation channel. Even then, however, component zone broadening is unavoidable during the separation process for the reasons stated above. By contrast, cIEF allows the whole separation channel to be filled with the analyte sample mixture without any deterioration of the separation resolution. In comparison to other CE modes, this provides cIEF with a much higher analyte loading capacity.
The absence of parabolic flow broadening with static, focused zones and low electropheresis current makes cIEF's separation resolution much less dependent on small dimensional separation channels. The separation channel's cross-sectional dimension can be 2 to 5 times larger than other CE modes with comparable separation resolution. Use of a larger cross-sectional separation channel again further increases sample analyte loading capacity. Higher sample loading provides a higher amount of extracted component material. Increasing the amount of extracted material is highly desirable since it generally increases the analytical success of subsequent analytical techniques such as mass spectrometry.
For the foregoing reasons, cIEF would appear to be an attractive technique for nano/micro preparative fractionation of closely related proteins from a mixture, for example, variants of hemoglobin arising from mutations to the amino acids sequences; or different forms of recombinant proteins arising from the heterogeneity associated with different post-translational modifications.
Others have investigated the use of cIEF for fraction collection, as an aspect of CE fractionation generally. For example, Guttman et al [4,5] have studied “planar” electrophoresis using a capillary cross-connector. By applying different voltage configurations through different reservoirs, a component zone or peak was collected after passing a single-point detector.
To date, however, others have had limited success in achieving any high degree of precision in the selection and extraction of cIEF component zones, chiefly because of the following limitations to the operation of their devices: 1) mobilization flow is in one direction; 2) mobilization speed is pre-determined and generally fixed and 3) inability to visualize the entire separation zones and detect in real-time any zone-width distortion due to mobilization.
In conventional “single point on-column” detection cIEF, the focused zones or peaks within the capillary must be moved, chemically or electroosmotically, past the detection point to be detected. This mobilization step in cIEF requires extra time and distorts the focused peaks, making it difficult to collect pure peaks without cross-contamination when peaks focus in close proximity.
It has, accordingly, not been possible with existing microfluidic (capillary) electrophoretic devices for micro-preparative fraction collection to observe all of the separated peaks developed by electrophoresis, then select a particular peak of interest and mobilize the entire pattern of peaks, while maintaining or re-establishing the focus of the pattern to bring the selected peak to a separation/collection point.
It is a principal object of the current invention to provide an integrated micro-scale electrophoresis device (IMED) for the extraction of large (μg) or small (ng) amounts of components (in particular, proteins) separated by cIEF.
It is a particular object of the invention to provide such an IMED as aforesaid which is adapted for automatic sample injection and capable of high-resolution protein separation, selection of a specific protein zone or peak and precision extraction of the central portion of the selected protein peak (“heart-cut extraction”) for further characterization.